Fig. 2

N-acylethanolamine binding receptors visualized by western blotting and immunofluorescence and quantification of CB₁, CB₂, TRPV1 and TRPA1 in response to hypoxia and TNF in RASF by cell-based ELISA. Immunofluorescent staining and western blotting revealed the cellular localization of CB₁ (a), CB₂ (b), TRPA1 (c), TRPV1 (d), FAAH (e) and COX-2 (f) protein. Original magnification is 400×. Quantification of CB₁ (g), CB₂ (h), TRPA1 (i) and TRPV1 (j) protein by cell-based ELISA after culture of RASF for 48 h under either hypoxia, TNF stimulation (10 ng/ml) or hypoxia combined with TNF (10 ng/ml) in RASF. g-j: Data are given as % of unstimulated control RASF. *p <0.05 vs. normoxic control (dotted line). Paired Wilcoxon test was used for comparisons. LY = complete cell lysate, CF = cytosolic fraction, NF = nuclear fraction, N = normoxia, H = hypoxia, PARP = poly(ADP-ribose)-polymerase 1 (control for nuclear localization), GAPDH = glycerinaldehyd-3-phosphat-dehydrogenase (control for cytosolic localization). CB ₁ cannabinoid receptor type 1, CB ₂ cannabinoid receptor type 2, COX-2 cyclooxygenase-2, ELISA enzyme-linked immunosorbent assay, FAAH fatty acid amide hydrolase, IL-6 interleukin 6, IL-8 interleukin 8, MMP-3 matrix metalloproteinase 3 (stromelysin), RA rheumatoid arthritis, SF synovial fibroblast(s), TNF tumor necrosis factor, TRPA1 transient receptor potential ankyrin 1, TRPV1 Transient receptor potential vanilloid channel